Our lab has been studying the role of signal transducer and activator of transcription 5 (STAT5) in hematopoiesis for more than a decade and we have identified important roles in hematopoietic and immune cell biology. Despite these advances in understanding the severity and broad range of defects in mice lacking STAT5, the mechanisms by which STAT5-mediated transcriptional activation controls hematopoiesis are not fully defined. We recently reported that although STAT5 drives multilineage differentiation it plays a surprising role in promoting hematopoietic stem cell quiescence. We have already identified key target genes involved in this process, including tie2 and p57. There are big differences between the hematopoietic stem cell and progenitor niches in regard to their oxidation potential and STAT5 is sensitive to reactive oxygen and nitrogen species. We will explore the overall hypothesis that STAT5-mediated transcriptional activation in the hypoxic hematopoietic stem cell niche is unique from that in the oxidation promoting hematopoietic progenitor niche and that changes in STAT5 function depend on the microenvironment and can control engraftment, mobilization, lineage differentiation, and quiescence. We have already performed preliminary gene expression array analysis of STAT5-deficient c-Kit+Sca-1+Lin- (KLS) cells and have utilized chromatin immunoprecipitation assays that identified bcl2 as a novel STAT5 direct target gene. Therefore we will test the following aims: 1) We will use a novel viable hematopoietic-specific conditional knockout mouse to study how STAT5 modulates juvenile seeding of the niches, migration, and mobilization. 2) We have demonstrated that the long-term repopulating hematopoietic stem cell pool is uniquely sensitive to STAT5 deletion, whereas the short-term repopulating pool is spared. We will analyze lineage differentiation in STAT5-deficient mice and characterize lymphoid priming and predisposition to B-lineage acute lymphoblastic leukemia. 3) We will perform STAT5 ChIP-qPCR experiments in multipotent hematopoietic cells and identify mechanisms of redox regulation. Collectively, these three aims will provide important insight into the functional and molecular regulation of hematopoiesis by STAT5 in response to early acting cytokines and may lead to improved therapeutic approaches for treating blood disorders.